Project Summary Our hypothesis is that Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) has a viral aetiology and that the characteristic persistent remitting and relapsing nature of the syndrome is the result of poorly controlled infection with one or more known viruses, possibly due to an underlying immune deficiency. We further hypothesise that the causative viruses will be human herpesviruses as they have the key characteristics of being highly prevalent, giving rise to lifelong infections, and undergoing prolonged periods of dormancy/latency with periodic reactivation at times of stress or immune compromise. Linking viral infections to ME/CFS has been difficult due to the cross-sectional nature of most epidemiological studies of this disease. Thus, although differences in prevalence of some viruses have been reported, causal inference is lacking. We believe that the development of highly sensitive, specific and quantitative nucleic acid based tests for quantification of human herpesviruses in non-invasive samples (e.g. saliva, urine), will allow us to conduct large scale prospective studies that will provide robust evidence for or against a link between herpesvirus reactivation and ME/CFS. The purpose of this R21 application is to conduct exploratory/ developmental work that will underpin future, large scale, epidemiological studies. Our specific objectives are: 1) To develop and validate highly sensitive and specific, quantitative digital droplet PCR (ddPCR) tests to detect all 8 human herpes viruses in blood and in more easily accessible biological samples (e.g. saliva, urine). 2) To use these assays to test samples from 250 ME/CFS cases, 200 healthy controls, and 50 controls with confirmed multiple sclerosis (MS), in the UK and Norwegian ME/CFS biobanks and look for associations between herpes virus infection, ME/CFS status and clinical parameters. 3) In a subset of virus positive and negative participants, to conduct a pilot study to monitor changes in herpesvirus viral load over time (monthly sampling) and correlate this with clinical and biological markers. This study is novel in that it will use highly sensitive and specific molecular assays to detect and very accurately quantify circulating viral loads from noninvasively collected samples. When combined with our ongoing recruitment and follow up of ME/CFS cases and controls in the UK (funded by 1R01AI103629-01A1) and our collaboration with the Norwegian ME/CFS biobank, this study will allow us, for the very first time, to correlate virus infection and reactivation with remission and relapse of clinical symptoms. If our hypotheses are found to be supported by the evidence, this will offer insights in to the underlying pathophysiology of the disease and open up new avenues for treatment and management of ME/CFS.